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soo, i've been digesting the info about shooms being polycaryotes and that they swap genetic material. and i have a thought that might look like an idea (lets see if i can put it into words).

if one has several monocultures on agar(from fruitbodies) that have desireable qualities. and one place samples of two of those on the same agarplate. then, as the mycs grow together, they would swap nuceleis with each other. so that transfering samples from the intersecting region one would get (one or several) cultures with genetic material from both the original samples. (which could then be spawned to see if its a desireable mix).

if this is so... it would be a very quick and simple way of cultivating desireable traits in a strain.

any thoughts?

--------------------Mindzpores words of wisdom:"If you think something is foolproof, you just haven't met proper fools".

Wiccan_Seeker said:"It is better to adjust to become a better listener than to keep on cranking up the volume".

My thoughts are that this is exactly what mushroom breeders do. It's the most obvious and basic breeding technique, constantly used by commercial strain producers as well as amateurs. It is, essentially, what everyone wanting to artifically breed a new strain of any sexual lifeform has done for thousands of years: mate parents with desirable qualities and then check out the offspring (although admittedly doing this at the level of the monokaryon is controlling selection at a highly refined, gametic level).

In any case, it's often more useful to isolate and test robust strains emerging from multispore inoculations, as these are composed of nuclear types that have already been selected for dominance and compatibility by merit of having outcompeted other types on the chosen medium.

Another thing to consider is that breeding together monokayons with desired traits to produce 'blended' dikaryons is tricky if these traits are related to fruiting characteristics. Specific fruiting qualities are emergent properties of specific dikaryoses and cannot readily be untangled into discrete inheritable factors of their component and offspring monokaryons.

By the way, when two monokaryons meet, fuse, and achieve reciprocal nuclear exchange, then they are both converted into the same dikaryon. There is no need to make isolations from the "intersecting region" as you put it (??) as the entire culture is now homogenously dikaryotic. Isolations from anywhere in this culture will yield identical subcultures in terms of nuclear composition. Of course, many other genetic outcomes are possible following strain interaction, just not in the exact scenario you describe above.

ok, i was just trying to get around the whole multispore selection bit as it seems the most time consuming and hard to control.

i thought that they would only exchange nucleatic material where they met. and did not realize that the myc would transfer and swap throughout the entire system. wow, thats even more crazy... but now with a method to it.

then it would be possible to blend several monocultures with desireable traits at one time.

so what you are saying is that basically, the most efficient way would be to use alternating cykles of selective agar mating and multispore selection.

blending monocultures in agar, then fruiting that, using its sporeprints for starting another cykle.

--------------------Mindzpores words of wisdom:"If you think something is foolproof, you just haven't met proper fools".

Wiccan_Seeker said:"It is better to adjust to become a better listener than to keep on cranking up the volume".

If you'll place two different dikaryotic strains on petri dishes a few hundred times, you'll probably get one or two dikaryotic pairings. In the vast majority of cases, a zone of isolation will develop between the two strains and that will be the end of it. Eventually, one strain may overrun the other, but no dikaryotic pairings take place. I've been attempting this for years, and have only two success stories so far. The hybrid in this instance will emerge from the zone of isolation between the two growths on the petri dish, and will be a distinct third sector. You can test your hybrid by placing a small sample of it and the two parent cultures on a third plate. If a zone of isolation develops between all three, you know have successfully created a hybrid. Stamets teaches this tek at his master's seminar.RR

hmmm... ok, thats bad news... how are those hybrids you created, do you find that they indeed show an improvement in performance compared to the original cultures, or is the difference to small to note?

would the rate of hybridizaton increace if one added more cultures to each dish?

also, im instantly thinking that there must be a chemical trigger that causes the "zone of isolation". so theoretically (while i can not, and its probably a huge and costly project) it would be possible to indentify that trigger and dose the agar with a chem that stops the isolation from occuring.

--------------------Mindzpores words of wisdom:"If you think something is foolproof, you just haven't met proper fools".

Wiccan_Seeker said:"It is better to adjust to become a better listener than to keep on cranking up the volume".

Mindzpore - yes, reciprocal nuclear exchange typically results in the complete dikaryotization of both parent monokaryons. The migrating nuclei can travel at speeds of up to four centimetres per hour(!). Migrating nuclei are seen as performing a male function and the accepting networks a female function. This phenomenon results in a uniform dikaryon that nevertheless can be a cytoplasmic chimera if the parental mitochondrial genotypes differ. To expand on this, trait differences between dikaryons having exactly the same nuclear composition can still be explained by genetic and epigenetic factors.

No, this system does not mean it is possible "to blend several monocultures with desireable traits at one time." Fusing more than two monokaryons together at once results in a multispore scenario from which a single or multiple dominant dikaryons arise, each composed of paired nuclei from two of the original monokaryons. There is no way of combining genetic information from more than two monokaryons into a dikaryon via straightforward mating. Unusual phenomena such as inequal nuclear ratios, partial migration, unidirectional migration, and aggresively invasive nuclear types have all been reported from various mushroom species, but certainly no *stable* effectively triploid or otherwise polyploid mushroom mycelia have been described in the genetics literature. Parasexual recombination could achieve genetic representation by more than two haplotypes in a tertiary dikaryon, but this phenomenon is unstudied in psilocybes and cannot be reliably manipulated in any mushroom species.

I agree with you that multispore selection is hard to control (in fact, absence of control is what makes this procedure useful), and is also hard to observe empirically, *but* it is hardly a time consuming process. You simply transfer a mass of spores onto agar and subculture the robust sectors which emerge over a few plates. Mating monokaryons is, on the other hand, *very* time consuming. Isolating a substantial number of individual germinating spores, running a large batch of crossing trials, and then quantifying the properties of the strains thus obtained.... hassle!!! I do not know many others within the amateur cultivation community who are interested in or capable of performing single spore isolations. I read a thread about failed between-strain P. cubensis matings once upon a time, but I cannot remember the details. It'd be great if someone picked up this work again; there is a lot to be learned from experimenting with P. cubensis strains at the monokaryon level.

You say that what I'm saying is "the most efficient way would be to use alternating cykles of selective agar mating and multispore selection." But I'm not saying that - it would be overgeneralizing. The most effective breeding regime would depend on precisely what you're selecting for and also on the relatedness of your breeding stock.

Finally, your idea about a chemical additive to overcome strain incompatibility somewhat underestimates the genetic complexity and hard-wiring of mushroom incompatibility systems. The mechanism directing formation of isolation zones is not a "chemical trigger" but a holistic developmental response co-ordinated by multiple genes, each with their own molecular recognition systems and subsequent activation pathways. Some types of incompatibility can be partially or wholly overcome by forcible methods (e.g. protoplast fusion), while others are impossible to circumvent without re-writing entire sections of the mushroom genome. So, a universal chemical additive to overcome incompatibility in general is fantasy, but chemicals to induce hybridization between incompatible strains in highly specific circumstances have already been developed. In any case, you do not need to try and force dikaryons to mate to produce hybrid strains (see below).

Roger, it's interesting you've been able to obtain new strains by interfacing dikaryons. I had observed this in several species but not psilocybes. Do you have anything interesting to report about matings between different strains? Have you worked with monokaryons?

Another more reliable - but more technically difficult - way of mating two dikaryons of a single species is by de-dikaryotizing the dikaryons (via physical and/or chemical fragmentation followed by protoplast regeneration) and mating the four recovered haplotypes. The number of possible offspring is then determined by the mating and mito- types of these monokaryons. To keep track of the parental composition of each hybrid, at least one molecular marker is necessary (PCR-RFLP has proved popular but is becoming superceded by newer methods). I have seen these techniques used in commercial Agaricus and Pleurotus breeding programmes. However, starting breeding regimes from a stock of monokaryons reduces the need for dikaryon hybridization and molecular tracking of their constituent haplotypes. Finally, I am unhappy with our use of the term 'hybridization' in the situations we've been discussing - as far as I know we've really just talking about newly generated dikaryons within a single species (would you say you are a 'hybrid' of your mother and father?).

"certainly no *stable* effectively triploid or otherwise polyploid mushroom mycelia have been described in the genetics literature."

but I had forgotten about certain experiments with Armillaria ostoyae and Armillaria gallica, showing that some matings of 2n + n strains result in an effective stable triploidy, and also that chimerism of multiple (as high as nine) nuclear types can occur for sustained periods in some wild mycelia and mushrooms. This latter scenario is almost certainly the result of post-fusion recombinatory interactions (i.e. parasexuality or re-haploidization) between two parental genotypes as opposed to simultaneous or sequential mating between multiple genotypes. Chimerism can persist in the same mycelium over many fruiting seasons.